At one time, it was thought that the debilitating, and often deadly, disease known generically as tuberculosis had been conquered as an anathema to mankind. Unfortunately, recent data indicates that the incidence of tuberculosis in humans has not been eradicated but is becoming more prevalent.
In treating humans afflicted with tuberculosis, the length of treatment and minimization of damage to the lungs is directly dependent upon early diagnosis of the condition. The earlier the diagnosis, the shorter the treatment term and the less the damage. Traditionally, it has been common medical practice to clinically diagnose tuberculosis in the lungs of a human being by a number of techniques including chest X-rays and cultures grown from sputum of the patient. Tuberculosis is a condition caused in the lungs by Mycobacterium tuberculosis. Regrettably, the tuberculosis condition in the lungs must be relatively well advanced before symptoms show up in the patient. Typical symptoms are coughing sputum and pains in the chest area. When the symptoms appear, diagnosis procedures are conducted clinically. Spots on the lung noted by chest X-rays, and clinical cultures developed in the laboratory, microscopic analysis of sputum of afflicted patients, are not reliable until the tuberculosis condition is relatively well advanced.
There is a strong need for a reliable procedure for early diagnosis of tuberculosis. Hopes of developing useful serodiagnostic tests for tuberculosis were heightened recently by the discovery of a Mycobacterium tuberculosis species-specific trehalose-containing glycolipid provisionally designated as SL-IV (Papa et al., 1989; Cruaud et al., 1990). This compound was originally thought to bear a sulphate ester group in position 2' (Daffe et al., 1990); however, a recently completed reinvestigation of the structure failed to confirm the presence of sulphate (Baer, personal communication). An ELISA serological procedure using this 2,3-O-diacyltrehalose has been shown to have good potential in the diagnosis of tuberculosis and leprosy (Cruaud et al., 1990).
Trehalose-based glycolipids are found in a variety of structural forms in the lipids of mycobacteria and related bacteria. Serologically active glycolipids extracted from M. bovis BCG have been described (Reggiardo and Middlebrook, 1975a,b). Three families of glycolipids called A, B and C reacted with sera from patients with tuberculosis and leprosy. Among the antigens studied, one designated A1 gave the lowest incidence of false-negative serologic reactions and was proved to be 6-o-mycoloyltrehalose or TMM (Reggiardo et al., reported in Goren, 1990). Cord factor (6,6'-di-o-mycoloyltrehalose (TDM)), on the other hand, does not seem to be useful as a coating antigen (Goren, 1990). Pseudo cord factors in which the carbon atoms C-6 and C-6' of the trehalose have been transformed either to carboxylic esters or to carboxamide functions, have been synthetized. Some of them, notably acylated dideoxydiamino derivatives, exhibit greater toxicity in mice than the simple cord factor analogue 6,6'-di-o-palmitoyltrehalose or the cord factor itself (Goren, 1990). A recent report shows that a neoglycoconjugate of bovine serum albumin (BSA) containing similar pseudo antigens was useful in the serodiagnosis of coccidioidomycosis (Goren et al., 1990). Among other biological characteristics, cord factors and their synthetic, albeit simpler analogues, induce granuloma formation in mice and have anti-tumour activity (Bekierkunst, 1984).
An article entitled "Evaluation of synthetic pseudo cord-factor-like glycolipids for the serodiagnosis of tuberculosis", Res. Microbiol. 1992, 143, 217-223, A. Laszlo et al., discloses five glycolipids which were evaluated in an ELISA test for their serodiagnostic usefulness in tuberculosis. One hundred and twelve (112) sera belonging to bacteriologically confirmed TB patients, patients with pathologies other than tuberculosis and healthy individuals were examined against several synthetic "mirror" pseudo cord factors (analogues of trehalose-6,6'-dimycolate or TDM) using natural cord factor and another recently described natural glycolipid (SL-IV) of Mycobacterium tuberculosis as control antigens. Analysis of the results showed that all synthetic "mirror" pseudo cord factors, except one with a short-8-carbon chain, were better recognized by the sera of tuberculosis patients than natural cord factor, with sensitivity and specificity values in the ELISA test similar to those reported for M. tuberculosis species-specific SL-IV.
Of all antigens tested in that study, BDA.TDA, a bis(N,N-dioctadecylamide) of "trehalose dicarboxylic acid", [(.alpha.-D-glucopyranosyluronic acid) (.alpha.-D-glucopyranosiduranic acid)], showed the highest serodiagnostic discriminating power (93% sensitivity and specificity). It was postulated that either these artificial molecules are cross-reactants of similarly structured native glycolipids of M. tuberculosis or that they bear closer resemblance to actual phagosome-lysosome-modified antigens than to native mycobacterial ones.
Seven antigens were tested, namely, five bisalkyl esters (I) of "homotrehalosuronic acid" (6-deoxy-.alpha.-D-gluco-heptopyranosyluronic acid, 6-deoxy-.alpha.-D-glucoheptopyranosiduronic acid) were synthesized as described (Baer and Breton, 1991; Baer et al., 1990). Differing in the length of their lipid chains R, they represent a novel type of "mirror" pseudo cord factor structure. The "mirror amide" pseudo cord factor (II) was synthesized. It is trehalose dicarboxylic acid bis (N,N-dioctadecylamide) (BDA.TDA). Cord factor (III, TDM) "Peurois" was originally obtained from the late Dr. E. Lederer. The SL-IV (Daffe et al., 1989; Cruaud et al., 1989) obtained from the Pasteur Institute, were used as natural M. tuberculosis antigens for reference.
Test data for the seven different antigens varied considerably. The sensitivity and specificity of the antigen showing the highest serodiagnostic discriminating power (93 percent), namely, BDA.TDA, a bis (N,N-dioctadecylamide) of trehalose dicarboxylic acid [(.alpha.-D-glucopyranosiduronic) (.alpha.-D-glucopyranosiduranic acid)] was more than double the bis-alkyl ester having the substituent n-C.sub.15 H.sub.31, pentadecyl, and almost three times the data for TDM, which incorporated the substituent mycoloyl. These data demonstrate that it is not possible to predict with any reliability the sensitivity and specificity of such antigens without conducting specific tests. The predictability factor in endeavouring to extrapolate the effectiveness of one antigen, based on homologues or similar organic structures, is minimal.